Mutant protein

ABSTRACT

The present invention relates to an immunoglobulin-binding protein, wherein at least one asparagine residue has been mutated to an amino acid other than glutamine or aspartic acid, which mutation confers an increased chemical stability at pH-values of up to about 13-14 compared to the parental molecule. The protein can for example be derived from a protein capable of binding to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), such as protein A, and preferably the B-domain of Staphylococcal protein A. The invention also relates to a matrix for affinity separation, which comprises an immunoglobulin-binding protein as ligand coupled to a solid support, in which protein ligand at least one asparagine residue has been mutated to an amino acid other than glutamine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/568,854 filed Sep. 29, 2009, now U.S. Pat. No. 8,354,510, which is adivisional of U.S. patent application Ser. No. 11/374,532 filed Mar. 13,2006, now abandoned, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/508,625 filed Sep. 20, 2004, now U.S. Pat. No.7,834,158, which is a filing under 37 U.S.C. §371 and claims priority tointernational patent application number PCT/SE03/00475 filed Mar. 20,2003, published on Oct. 2, 2003 as WO03/080655 and also claims priorityto patent application number 0200943-9 filed in Sweden on Mar. 25, 2002;the disclosures of which are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present invention relates to the field of mutant proteins, and morespecifically to a mutant protein that exhibits improved stabilitycompared to the parental molecule as well as to a method of producing amutant protein according to the invention. The invention also relates toan affinity separation matrix, wherein a mutant protein according to theinvention is used as an affinity ligand.

BACKGROUND OF THE INVENTION

A great number of applications in the biotechnological andpharmaceutical industry require comprehensive attention to definiteremoval of contaminants. Such contaminants can for example be non-elutedmolecules adsorbed to the stationary phase or matrix in achromatographic procedure, such as non-desired biomolecules ormicroorganisms, including for example proteins, carbohydrates, lipids,bacteria and viruses. The removal of such contaminants from the matrixis usually performed after a first elution of the desired product inorder to regenerate the matrix before subsequent use. Such removalusually involves a procedure known as cleaning-in-place (CIP), whereinagents capable of eluting contaminants from the stationary phase areused. One such class of agents often used is alkaline solutions that arepassed over said stationary phase. At present the most extensively usedcleaning and sanitizing agent is NaOH, and the concentration thereof canrange from 0.1 up to e.g. 1 M, depending on the degree and nature ofcontamination. NaOH is known to be an effective CIP agent achievingmultilog reduction of contaminants, such as microbes, proteins, lipidsand nucleic acids. Another advantage of NaOH is that it can easily bedisposed of without any further treatment. However, this strategy isassociated with exposing the matrix for pH-values above 13. For manyaffinity chromatography matrices containing proteinaceous affinityligands such alkaline environment is a very harsh condition andconsequently results in decreased capacities owing to instability of theligand to the high pH involved.

An extensive research has therefore been focussed on the development ofengineered protein ligands that exhibit an improved capacity towithstand alkaline pH-values. For example, Gülich et al (Susanne Gülich,Martin Linhult, Per-Åke Nygren, Mathias Uhlén, Sophia Hober, Journal ofBiotechnology 80 (2000), 169-178: Stability towards alkaline conditionscan be engineered into a protein ligand) suggested protein engineeringto improve the stability properties of a Streptococcal albumin-bindingdomain (ABD) in alkaline environments. Previously, it was shown thatstructural modification, such as deamidation and cleavage of the peptidebackbone, of asparagine and glutamine residues in alkaline conditions isthe main reason for loss of activity upon treatment in alkalinesolutions, and that asparagine is the most sensitive of the two (Geiger,T., and S. Clarke. 1987. Deamidation, Isomerization, and Racemization atAsparaginyl and Aspartyl Residues in Peptides. J. Biol. Chem.262:785-794). It is also known that the deamidation rate is highlyspecific and conformation dependent (Kosky, A. A., U. O. Razzaq, M. J.Treuheit, and D. N. Brems. 1999. The effects of alpha-helix on thestability of Asn residues: deamidation rates in peptides of varyinghelicity. Protein Sci. 8:2519-2523; Kossiakoff, A. A. 1988. Tertiarystructure is a principal determinant to protein deamidation. Science.240:191-194; and Lura, R., and V. Schirch. 1988. Role of peptideconformation in the rate and mechanism of deamidation of asparaginylresidues. Biochemistry. 27:7671-7677), and the shortest deamidation halftimes have been associated with the sequences—asparagine-glycine- and-asparagine-serine. Accordingly, Gülich et al created a mutant of ABD,wherein all the four aspargine residues of native ABD have been replacedby leucine (one residue), asparte (two residues) and lysine (oneresidue).

Further, Gülich et al report that their mutant exhibits a target proteinbinding behaviour similar to that of the native protein, and thataffinity columns containing the engineered ligand show higher bindingcapacities after repeated exposure to alkaline conditions than columnsprepared using the parental non-engineered ligand. Thus, it is concludedtherein that all four asparagine residues can be replaced without anysignificant effect on structure and function.

Thus, the studies performed by Gülich et al were performed on aStreptococcal albumin-binding domain. However, affinity chromatographyis also used in protocols for purification of other molecules, such asimmunoglobulins, e.g. for pharmaceutical applications. A particularlyinteresting class of affinity reagents is proteins capable of specificbinding to invariable parts of an antibody molecule, such interactionbeing independent on the antigen-binding specificity of the antibody.Such reagents can be widely used for affinity chromatography recovery ofimmunoglobulins from different samples such as but not limited to serumor plasma preparations or cell culture derived feed stocks. An exampleof such a protein is staphylococcal protein A, containing domainscapable of binding to the Fc and Fab portions of IgG immunoglobulinsfrom different species.

Staphylococcal protein A (SpA) based reagents have due to their highaffinity and selectivity found a widespread use in the field ofbiotechnology, e.g. in affinity chromatography for capture andpurification of antibodies as well as for detection. At present,SpA-based affinity medium probably is the most widely used affinitymedium for isolation of monoclonal antibodies and their fragments fromdifferent samples including industrial feed stocks from cell cultures.Accordingly, various matrices comprising protein A-ligands arecommercially available, for example, in the form of native protein A(e.g. Protein A SEPHAROSE™, Amersham Biosciences, Uppsala, Sweden) andalso comprised of recombinant protein A (e.g. rProtein A SEPHAROSE™,Amersham Biosciences, Uppsala, Sweden). More specifically, the geneticmanipulation performed in said commercial recombinant protein A productis aimed at facilitating the attachment thereof to a support.

Accordingly, there is a need in this field to obtain protein ligandscapable of binding immunoglobulins, especially via the Fc-fragmentsthereof, which are also tolerant to one or more cleaning proceduresusing alkaline agents.

BRIEF SUMMARY OF THE INVENTION

One object of the present invention is to provide a mutatedimmunoglobulin-binding protein ligand that exhibits an improvedstability at increased pH-values, and accordingly an improved toleranceto cleaning under alkaline conditions, as compared to the parentalmolecule.

Another object of the invention is to provide such a protein ligand,which binds specifically to the Fc-fragment of immunoglobulins, such asIgG, IgA and/or IgM.

Yet another object of the invention is to provide a protein ligand asdescribed above, which also exhibits an affinity which is retained for alonger period of time in alkaline conditions than that of the parentalmolecule.

A further object of the present invention is to provide an affinityseparation matrix, which comprises mutant protein ligands capable ofbinding immunoglobulins, such as IgG, IgA and/or IgM, preferably viatheir Fc-fragments, which ligands exhibit an improved tolerance tocleaning under alkaline conditions, as compared to the parental moleculeligand.

One or more of the above-defined objects can be achieved as described inthe appended claims.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NOs: 5-9, 1 & 10-11, respectively, in order ofappearance) shows amino acid alignments of the five homologous domains(E, D, A, B and C) of SpA. Horizontal lines indicate amino acididentity. The three boxes show the α-helices of Z_(wt) as determined byTashiro and co-workers (Tashiro et al., 1997). The asparagine residues,and also one glycine residue in the B domain, which were replaced, areunderlined in the figure. Also, amino acid alignments for Zwt andZ(N23T) are shown.

FIGS. 2( a) and 2(b) illustrate the results obtained after alkalinetreatment (cleaning-in-place) of mutant proteins according to theinvention as compared to the destabilized protein Z. A comparison of thecapacity after repeated CIP-treatment following an ordinary affinitychromatography scheme. 0.5 M NaOH was used as cleaning agent. Theprotocol was run 16 times and the duration for the alkaline sanitizationwas 30 minutes in each round. FIG. 2( a) shows the inactivation patternfor Z(F30A) and variants thereof, whereas FIG. 2( b) shows theinactivation pattern for Zwt and Z(N23T).

FIG. 3 (SEQ ID NOs: 12-13) shows the gene encoding theZ(N23T/N3A/N6D)-Cys after insertion into vector as described in example4(a). The mutations are marked with *.

FIG. 4 shows a plasmid map of the plasmid pAY91, which contains the geneencoding Z(N23T/N3A/N6D)-Cys as described in example 4(a).

FIG. 5 (SEQ ID NOs: 14-15) shows the gene encoding the Z(N23T/N3A/N6D)after insertion into vector as described in example 4(b). The mutationsare marked with *.

FIG. 6 shows an example of plasmid map for the plasmid pAY100 expressingthe tetramer of Z(N23T/N3A/N6D)-Cys as described in example 5.

FIG. 7 (SEQ ID NOs: 16-17) shows the adapter for introducing a KpnI-siteinto a vector with SPA promoter and signal sequence according to example6.

FIG. 8 shows the plasmid pAY104, which contains SPA promoter and signalsequence to be used for introduction of an adapter containing aKpnI-site, as described in example 6.

FIG. 9 shows the resulting plasmid, pAY128, after insertion of theadapter according to example 6.

FIG. 10 (SEQ ID NO: 18) shows the constructed cloning cassette ofexample 6, where the original adapter is underlined.

FIG. 11 shows plasmid pAY114 after insertion of theZ(N23T/N3A/N6D)-Cys-tetramer as described in Example 6.

FIG. 12 (SEQ ID NOs: 19-21, respectively, in order of appearance) showsthe constructed cloning cassette of example 7, where the originaladapter is underlined.

FIG. 13 shows the resulting plasmid, pAY129, after insertion of theadapter according to example 7.

FIG. 14 shows plasmid pAY125 after insertion of theZ(N23T/N3A/N6D)tetramer-Cys as described in example 7.

FIG. 15 is a chromatogram obtained from a run as described in example 8,where the first peak corresponds to the flow-through material and thesecond peak corresponds to eluted hIgG.

FIG. 16 shows graphs that represent the remaining dynamic bindingcapacity of the matrices in accordance with example 8. From top tobottom they represent Z(N23T/N3A/N6D)dimer-Cys, Z(N23T/N3A)dimer-Cys,Z(N23T)dimer-Cys and Z(N23T/K4G)dimer-Cys respectively. Due to softwareproblems the last two measure points for Z(N23T/N3A)dimer-Cys arelacking.

FIG. 17 shows the results of Experiment 1 as described in Example 9below, wherein HERCEPTIN®, a monoclonal antibody which comprises VH3, isseparated on 4 different matrices as described in the Experimental partbelow.

FIG. 18 shows the results of Experiment 1 as described in Example 9below, wherein ENBREL™, a recombinant protein, is separated on the same4 matrices.

FIG. 19 shows the results of Experiment 1 as described in Example 9below, wherein the monoclaonal antibody SYNAGIS® is separated on 4different matrices.

FIG. 20 shows the results of Experiment 2 as described in Example 9below, wherein HERCEPTIN® is separated on 4 different matrices asdescribed in the Experimental part below.

FIG. 21 shows the results of Experiment 2 as described in Example 9below, wherein ENBREL™ is separated on 4 separation matrices.

FIG. 22 shows the results of Experiment 2 as described in Example 9below, wherein SYNAGIS® is separated on 4 different separation matrices.

DEFINITIONS

The term “protein” is used herein to describe proteins as well asfragments thereof. Thus, any chain of amino acids that exhibits a threedimensional structure is included in the term “protein”, and proteinfragments are accordingly embraced.

The term “functional variant” of a protein means herein a variantprotein, wherein the function, in relation to the invention defined asaffinity and stability, are essentially retained. Thus, one or moreamino acids that are not relevant for said function may have beenexchanged.

The term “parental molecule” is used herein for the correspondingprotein in the form before a mutation according to the invention hasbeen introduced.

The term “structural stability” refers to the integrity ofthree-dimensional form of a molecule, while “chemical stability” refersto the ability to withstand chemical degradation.

The term “Fc fragment-binding” protein means that the protein is capableof binding to the Fc fragment of an immunoglobulin. However, it is notexcluded that an Fc fragment-binding protein also can bind otherregions, such as Fab regions of immunoglobulins.

In the present specification, if not referred to by their full names,amino acids are denoted with the conventional one-letter symbols.

Mutations are defined herein by the number of the position exchanged,preceded by the wild type or non-mutated amino acid and followed by themutated amino acid. Thus, for example, the mutation of an asparagine inposition 23 to a threonine is denoted N23T.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the preparation and use ofprotein-based ligands, often denoted affinity ligands. The presentinvention relates to a method of preparing a chromatography matrixcomprising at least one ligand with affinity for the Fc part of anantibody, which method comprises

-   (a) providing a nucleic acid sequence encoding at least one    alkali-stable domain B of staphylococcal Protein A (SpA);-   (b) mutating said nucleic acid sequence to encode for a recombinant    protein wherein at least one glycine has been replaced by alanine;-   (c) expressing the protein encoded by the nucleic acid sequence    resulting from (b) in a host cell; and-   (d) coupling the expressed protein to a support,    the improvement being that the ligand(s) so prepared lack affinity    for the Fab part of an antibody. In one embodiment, the ligand(s) so    prepared lack any substantial affinity for the Fab part of an    antibody.

In one embodiment of the present method, in the alkali-stable domain B,the alkali-stability has been achieved by mutating at least oneasparagine residue to an amino acid other than glutamine. In anadvantageous embodiment, the alkali-stability of domain B has beenachieved by mutating at least one asparagine residue to an amino acidother than glutamine; and (b) is a mutation of the amino acid residue atposition 29 of the alkali-stable domain B. Thus, in the last embodiment,the mutation is a G29A mutation. The numbering used herein of the aminoacids is the conventionally used in this field, and the skilled personin this field can easily recognize the position to be mutated.

In another embodiment, the recombinant protein expressed in (c) isProtein Z in which the alkali-stability has been achieved by mutating atleast one asparagine residue to an amino acid other than glutamine. Inan advantageous embodiment, the recombinant protein expressed in (c) isProtein Z in which the alkali-stability has been achieved by mutating atleast the asparagine residue at position 23 to an amino acid other thanglutamine. In another embodiment, the alkali-stable protein is a nativeprotein which is substantially stable at alkaline conditions.

As the skilled person in this field will easily understand, themutations to provide alkaline-stability and the G to A mutation of (b)may be carried out in any order of sequence. Consequently, the methodaccording to the invention embraces a method wherein an alkali-stableprotein is mutated from G to A; as well as the equivalent methodcomprising to first provide a nucleic acid encoding a G to A mutatedprotein which sequence is subsequently mutated to providealkali-stability.

Thus, in one embodiment, the present invention is a method of preparinga separation matrix comprising at least one ligand with affinity for theFc part of an antibody, which method comprises

-   (a) providing a nucleic acid sequence encoding a recombinant domain    B of staphylococcal Protein A (SpA), such as Protein Z;-   (b) mutating said nucleic acid sequence to encode for a recombinant    protein wherein at least one asparagine has been replaced by an    amino acid other than glutamine;-   (c) expressing the protein encoded by the nucleic acid sequence    resulting from (b) in a host cell; and-   (d) coupling the expressed protein to a support,    the improvement being that the ligand(s) so prepared lack affinity    for the Fab part of an antibody. In one embodiment, the ligand(s) so    prepared lack any substantial affinity for the Fab part of an    antibody.

Further, as the skilled person will understand, a protein having theadvantageous properties disclosed herein, such allowing elution ofantibodies at relatively high pH values when used in chromatography, maybe prepared by other methods than expression in host cells. Thus, oneaspect of the invention is a method of preparing a separation matrixcomprising at least one ligand with affinity for the Fc part of anantibody, which method comprises

-   (a) providing a protein ligand having the amino acid sequence of an    alkali-stable protein as discussed above, wherein at least one    asparagine has been replaced by an amino acid other than glutamine;    and-   (b) coupling the expressed protein to a support,    the improvement being that the ligand(s) so prepared lack affinity    for the Fab part of an antibody. In one embodiment, the ligand(s) so    prepared lack any substantial affinity for the Fab part of an    antibody. In an advantageous embodiment, the protein is provided    according to (a) by protein synthesis. Methods for synthesizing    peptides and proteins of predetermined sequences are well known and    commonly available in this field.

Thus, in the present invention, the term “alkali-stable domain B ofStaphylococcal Protein A” means an alkali-stabilized protein based onDomain B of SpA, such as the mutant protein described in the presentpatent application; as well as other alkali-stable proteins of otherorigin but having a functionally equivalent amino acid sequence.

Methods of providing mutations such as disclosed herein are well knownin this field and easily carried out by the skilled person followingstandard procedures. As the skilled person will understand, theexpressed protein should be purified to an appropriate extent beforebeing immobilized to a support. Such purification methods are well knownin the field, and the immobilization of protein-based ligands tosupports is easily carried out using standard methods. Suitable methodsand supports will be discussed below in more detail.

In an advantageous embodiment of the present method, the nucleic acidprovided encodes a multimer of two or more domains wherein at least oneis Protein Z in which the alkali-stability has been achieved by mutatingat least one asparagine residue to an amino acid other than glutamine.In an especially advantageous embodiment, the nucleic acid encodes arecombinant protein comprising two, three, four or five such domains,preferably combined by suitable linker elements.

In another aspect, the present invention relates to a method ofseparating antibodies, preferably monoclonal antibodies, from a liquid.Such separation is preferably a process of purifying antibodies bychromatography. Thus, in one embodiment of this aspect, the method is amethod of separating one or more antibodies from a liquid, which methodcomprises

-   (a) contacting the liquid with a separation matrix comprising    ligands immobilised to a support;-   (b) allowing antibodies to adsorb to the matrix by interaction with    the ligands;-   (c) an optional step of washing the adsorbed antibodies;-   (d) recovering antibodies by contacting the matrix with an eluent    which releases the antibodies;    the improvement being that ligands comprise one or more    alkali-stable domain B of staphylococcal Protein A (SpA) which have    been mutated to encode for a recombinant protein wherein at least    one glycine has been replaced by an alanine. In this context, it is    understood that the term “antibodies” embraces fusions comprising an    antibody portion as well as antibody fragments and mutated    antibodies, as long as they have substantially maintained the    binding properties of an antibody.

The conditions for the adsorption step may be any conventionally used,appropriately adapted depending on the properties of the target antibodysuch as the pI thereof. The elution may be performed by using anycommonly used buffer. In an advantageous embodiment, the recovery ofantibodies is achieved by adding an eluent having a pH in the range of3.6-4.0, preferably 3.7-3.9. In one embodiment, the elution pH is3.7±0.1. Thus, an advantage of this embodiment is that the targetantibody is exposed to pH values during elution which are as a rulehigher than conventionally used with protein A-based ligands, which formost antibodies will result in less degradation caused by reduced pH.

The present method is useful to capture target antibodies, such as afirst step in a purification protocol of antibodies which are e.g. fortherapeutic or diagnostic use. In one embodiment, at least 75% of theantibodies are recovered. In an advantageous embodiment, at least 80%,such as at least 90%, and preferably at least 95% of the antibodies arerecovered using an eluent having a pH in the range of 3.8-3.9. Thepresent method may be followed by one or more additional steps, such asother chromatography steps. Thus, in a specific embodiment, more thanabout 98% of the antibodies are recovered.

Further details and embodiments of the separation matrix provided in (a)may be as discussed above. In a specific embodiment, the separationmatrix used in the present method is MABSELECT™ SURE™ (GE Healthcare,Uppsala, Sweden). Thus, the invention also relates to the use ofMABSELECT™ SURE™ in chromatography wherein the elution pH is increasedas compared to what is conventionally used for Protein A-based ligands.

In one aspect, the present invention relates to animmunoglobulin-binding protein capable of binding to other regions ofthe immunoglobulin molecule than the complementarity determining regions(CDR), wherein at least one asparagine residue of a parentalimmunoglobulin-binding protein has been mutated to an amino acid otherthan glutamine, which mutation confers an increased chemical stabilityat alkaline pH-values compared to the parental molecule. The increasedstability means that the mutated protein's initial affinity forimmunoglobulin is essentially retained for a prolonged period of time,as will be discussed below.

The retained affinity for the target protein achieved according to theinvention is in part due to a retained spatial conformation of themutant protein. The affinity of mutated proteins to immunoglobulins canfor example be tested by the skilled person using biosensor technologyusing for example a BIACORE™ 2000 standard set-up (Biacore AB, Uppsala,Sweden), as will be illustrated in the experimental part below. In thiscontext, it is understood from the term “essentially” retained that themutated protein exhibits an affinity for immunoglobulin which is of thesame order of magnitude as that of the parental molecule. Accordingly,in an initial phase, the binding capacity of the mutated protein iscomparable with that of the parental molecule. However, due to thebelow-discussed chemical stability of the mutated protein, which isretained in time, its binding capacity will decrease more slowly thanthat of the parental molecule in an alkaline environment. Theenvironment can be defined as alkaline, meaning of an increasedpH-value, for example above about 10, such as up to about 13 or 14, i.e.from 10-13 or 10-14, in general denoted alkaline conditions.Alternatively, the conditions can be defined by the concentration ofNaOH, which can be up to about 1.0 M, such as 0.7 M or specificallyabout 0.5 M, accordingly within a range of 7-1.0 M.

The increased chemical stability of the mutated protein according to theinvention can easily be confirmed by the skilled person in this fielde.g. by routine treatment with NaOH at a concentration of 0.5 M, as willbe described in the experimental part below. In this context, it is tobe understood that similar to what is said above, an “increased”stability means that the initial stability is retained during a longerperiod of time than what is achieved by the parental molecule. Eventhough similar mutations have been reported for a Streptococcalalbumin-binding domain (Gülich et al, see above), it is well known thatthe rate of the deamidation involved in protein susceptibility todegradation in alkaline environments is highly sequence and conformationdependent. Since the amino acid sequence of ABD comprises no amino acidsequence similarity to immunoglobulin-binding proteins such as theindividual domains staphylococcal protein A, it would not appear asthough the teachings of Gülich et al could be applied also toimmunoglobulin-binding proteins. However, the present invention showsfor the first time that mutation of one or more asparagine residues ofan immunoglobulin-binding protein surprisingly provides an improvedchemical stability and hence a decreased degradation rate inenvironments wherein the pH is above about 10, such as up to about 13 or14.

Thus, the present invention provides a mutated protein, which is usefule.g. as a protein ligand in affinity chromatography for selectiveadsorption of immunoglobulins, such as IgG, IgA and/or IgM, preferablyIgG, from a mammalian species, such as a human. The purpose of theadsorption can be either to produce a purified product, such as a pureimmunoglobulin fraction or a liquid from which the immunoglobulin hasbeen removed, or to detect the presence of immunoglobulin in a sample.The ligand according to the invention exhibits a chemical stabilitysufficient to withstand conventional alkaline cleaning for a prolongedperiod of time, which renders the ligand an attractive candidate forcost-effective large-scale operation where regeneration of the columnsis a necessity.

Accordingly, in the protein according to the invention, one or moreasparagine (N) residues have been mutated to amino acids selected fromthe group that consists of glycine (G), alanine (A), valine (V), leucine(L), isoleucine (I), serine (S), threonine (T), cysteine (C), methionine(M), phenylalanine (F), tyrosine (Y), tryptophan (W), glutamic acid (E),arginine (R), histidine (H), lysine (K) or proline (P), or any modifiedamino acid that is not susceptible to the undesired deamidation andisomerisation. Alternatively, one or more asparagine (N) residues havebeen mutated to glutamine (Q).

The immunoglobulin-binding protein can be any protein with a nativeimmunoglobulin-binding capability, such as Staphylococcal protein A(SpA) or Streptococcal protein G (SpG). For a review of other suchproteins, see e.g. Kronvall, G., Jonsson, K. Receptins: a novel term foran expanding spectrum of natural and engineered microbial proteins withbinding properties for mammalian proteins, J. Mol. Recognit. 1999January-February; 12(1):38-44. Review.

In one embodiment, the present invention is a mutated protein, whichcomprises at least the binding region of an immunoglobulin-bindingprotein and wherein at least one such asparagine mutation is presentwithin said region. Accordingly, in this embodiment, a mutated proteinaccording to the invention comprises at least about 75%, such as atleast about 80% or preferably at least about 95%, of the sequence asdefined in SEQ ID NOs: 1 or 2, with the proviso that the asparaginemutation is not in position 21.

In the present specification, SEQ ID NO: 1 defines the amino acidsequence of the B-domain of SpA and SEQ ID NO: 2 defines a protein knownas protein Z. Protein Z is synthetic construct derived from the B-domainof SpA, wherein the glycine in position 29 has been exchanged foralanine, and it has been disclosed in the literature, see e.g. Stahl etal, 1999: Affinity fusions in biotechnology: focus on protein A andprotein G, in The Encyclopedia of Bioprocess Technology: Fermentation,Biocatalysis and Bioseparation. M. C. Fleckinger and S. W. Drew,editors. John Wiley and Sons Inc., New York, 8-22. Further, protein Zhas been used both as a ligand in affinity chromatography. However, eventhough protein Z exhibits an improved chemical stability to certainchemicals other than NaOH as compared to the SpA B-domain, it is stillnot as stable in conditions of increased pH-values as required towithstand the many CIP regeneration steps desired in an economicindustrial plant.

In one embodiment, the above described mutant protein is comprised ofthe amino acid sequence defined in SEQ ID NOs: 1 or 2, or is afunctional variant thereof. The term “functional variant” as used inthis context includes any similar sequence, which comprises one or morefurther variations in amino acid positions that have no influence on themutant protein's affinity to immunoglobulins or its improved chemicalstability in environments of increased pH-values.

In an advantageous embodiment, the present mutation(s) are selected fromthe group that consists of N23T; N23T and N43E; N28A; N6A; N11S; N11Sand N23T; and N6A and N23T; and wherein the parental molecule comprisesthe sequence defined by SEQ ID NO: 2. As mentioned above, in order toachieve a mutant protein useful as a ligand with high binding capacityfor a prolonged period of time in alkaline conditions, mutation of theasparagine residue in position 21 is avoided. In one embodiment, theasparagine residue in position 3 is not mutated.

In the most advantageous embodiment, in the present protein, anasparagine residue located between a leucine residue and a glutamineresidue has been mutated, for example to a threonine residue. Thus, inone embodiment, the asparagine residue in position 23 of the sequencedefined in SEQ ID NO: 2 has been mutated, for example to a threonineresidue. In a specific embodiment, the asparagine residue in position 43of the sequence defined in SEQ ID NO: 2 has also been mutated, forexample to a glutamic acid. In the embodiments where amino acid number43 has been mutated, it appears to most advantageously be combined withat least one further mutation, such as N23T.

The finding according to the invention that the various asparagineresidues of the B-domain of SpA and protein Z can be ascribed differentcontributions to affinity and stability properties of the mutatedprotein was quite unexpected, especially in view of the above discussedteachings of Gülich et al wherein it was concluded that all theasparagine residues of ABD could be mutated without any internaldiscrimination.

Thus, the invention encompasses the above-discussed monomeric mutantproteins. However, such protein monomers can be combined into multimericproteins, such as dimers, trimers, tetramers, pentamers etc.Accordingly, another aspect of the present invention is a multimercomprised of at least one of the mutated proteins according to theinvention together with one or more further units, preferably alsomutant proteins according to the invention. Thus, the present inventionis e.g. a dimer comprised of two repetitive units.

In one embodiment, the multimer according to the invention comprisesmonomer units linked by a stretch of amino acids preferably ranging from0 to 15 amino acids, such as 5-10. The nature of such a link shouldpreferably not destabilise the spatial conformation of the proteinunits. Furthermore, said link should preferably also be sufficientlystable in alkaline environments not to impair the properties of themutated protein units.

In the best embodiment at present, the multimer is a tetramer of proteinZ comprising the mutation N23T, wherein the length of the linking unitsare 5-10 amino acids. In one embodiment, the present multimer comprisesthe sequence VDAKFN-Z(N23T)-QAPKVDAKFN-Z(N23T)QAPKC (SEQ ID NO: 22). Inanother embodiment, the multimer comprises the sequenceVDAKFD-Z(N23T)-QAPKVDAKFD-Z(N23T)-ZQAPKC (SEQ ID NO: 23).

In a specific embodiment, the present multimer also comprises one ormore of the E, D, A, B, and C domains of Staphylococcal protein A. Inthis embodiment, it is preferred that asparagine residues located inloop regions have been mutated to more hydrolysis-stable amino acids. Inan embodiment advantageous for structural stability reasons, the glycineresidue in position 29 of SEQ ID NO: 1 has also been mutated, preferablyto an alanine residue. Also, it is advantageous for the structuralstability to avoid mutation of the asparagine residue in position 52,since it has been found to contribute to the α-helical secondarystructure content of the protein A molecule.

In a further aspect, the present invention relates to a nucleic acidencoding a mutant protein or multimer as described above. Accordingly,the invention embraces a DNA sequence that can be used in the productionof mutant protein by expression thereof in a recombinant host accordingto well-established biotechnological methods. Consequently, anotheraspect of the present invention is an expression system, which enablesproduction of a mutant protein as described above. Bacterial hosts canconveniently be used, e.g. as described in the experimental part below.In an alternative embodiment, the present invention is a cell line thathas been genetically manipulated to express a mutant protein accordingto the invention. For methods to this end, see e.g. Sambrook et al.,Molecular Cloning: A Laboratory Manual (2^(nd) ed), vols. 1-3, ColdSpring Harbor Laboratory, (1989).

Naturally, once the desired sequence has been established, the mutantprotein according to the invention can alternatively be produced bysynthetic methods.

Accordingly, the present invention also includes a biotechnological orsynthetic method of producing a mutant protein or a multimer accordingto the invention.

In another aspect, the present invention relates to a matrix foraffinity separation, which matrix comprises ligands that compriseimmunoglobulin-binding protein coupled to a solid support, in whichprotein at least one asparagine residue has been mutated to an aminoacid other than glutamine. The present matrix, when compared to a matrixcomprised of the parental molecule as ligand, exhibits an increasedbinding capacity during two or more separations with intermittentalkaline cleaning. The mutated protein ligand is preferably anFc-fragment-binding protein, and can be used for selective binding ofIgG, IgA and/or IgM, preferably IgG.

The matrix according to the invention can comprise the mutant protein asdescribed above in any embodiment thereof as ligand. In the mostpreferred embodiment, the ligands present on the solid support comprisea multimer as described above.

The solid support of the matrix according to the invention can be of anysuitable well-known kind. A conventional affinity separation matrix isoften of organic nature and based on polymers that expose a hydrophilicsurface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy(—COOH), carboxamido (—CONH₂, possibly in N-substituted forms), amino(—NH₂, possibly in substituted form), oligo- or polyethylenoxy groups ontheir external and, if present, also on internal surfaces. In oneembodiment, the polymers may, for instance, be based on polysaccharides,such as dextran, starch, cellulose, pullulan, agarose etc, whichadvantageously have been cross-linked, for instance with bisepoxides,epihalohydrins, 1,2,3-trihalo substituted lower hydrocarbons, to providea suitable porosity and rigidity. In the most preferred embodiment, thesolid support is porous agarose beads. The supports used in the presentinvention can easily be prepared according to standard methods, such asinverse suspension gelation (S Hjertén: Biochim Biophys Acta 79(2),393-398 (1964). Alternatively, the base matrices are commerciallyavailable products, such as SEPHAROSE™ FF (Amersham Biosciences,Uppsala, Sweden). In an embodiment, which is especially advantageous forlarge-scale separations, the support has been adapted to increase itsrigidity, and hence renders the matrix more suitable for high flowrates.

Alternatively, the solid support is based on synthetic polymers, such aspolyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides etc. In case ofhydrophobic polymers, such as matrices based on divinyl andmonovinyl-substituted benzenes, the surface of the matrix is oftenhydrophilised to expose hydrophilic groups as defined above to asurrounding aqueous liquid. Such polymers are easily produced accordingto standard methods, see e.g. “Styrene based polymer supports developedby suspension polymerization” (R Arshady: Chimica e L'Industria 70(9),70-75 (1988)). Alternatively, a commercially available product, such asSOURCE™ (Amersham Biosciences, Uppsala, Sweden) is used.

In another alternative, the solid support according to the inventioncomprises a support of inorganic nature, e.g. silica, zirconium oxideetc.

In yet another embodiment, the solid support is in another form such asa surface, a chip, capillaries, or a filter.

As regards the shape of the matrix according to the invention, in oneembodiment the matrix is in the form of a porous monolith. In analternative embodiment, the matrix is in beaded or particle form thatcan be porous or non-porous. Matrices in beaded or particle form can beused as a packed bed or in a suspended form. Suspended forms includethose known as expanded beds and pure suspensions, in which theparticles or beads are free to move. In case of monoliths, packed bedand expanded beds, the separation procedure commonly followsconventional chromatography with a concentration gradient. In case ofpure suspension, batch-wise mode will be used.

The ligand may be attached to the support via conventional couplingtechniques utilising, e.g. amino and/or carboxy groups present in theligand. Bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS)etc are well-known coupling reagents. Between the support and theligand, a molecule known as a spacer can be introduced, which willimprove the availability of the ligand and facilitate the chemicalcoupling of the ligand to the support. Alternatively, the ligand may beattached to the support by non-covalent bonding, such as physicaladsorption or biospecific adsorption.

In an advantageous embodiment, the present ligand has been coupled tothe support by thioether bonds. Methods for performing such coupling arewell-known in this field and easily performed by the skilled person inthis field using standard techniques and equipment. In an advantageousembodiment, the ligand is firstly provided with a terminal cysteineresidue for subsequent use in the coupling. The skilled person in thisfield also easily performs appropriate steps of purification.

As mentioned above, the affinity to immunoglobulin i.e. the bindingproperties of the present ligand, and hence the capacity of the matrix,is not essentially changed in time by treatment with an alkaline agent.Conventionally, for a cleaning in place treatment of an affinityseparation matrix, the alkaline agent used is NaOH and the concentrationthereof is up to 0.75 M, such as 0.5 M.

Thus, another way of characterising the matrix according to theinvention is that due to the above discussed mutations, its bindingcapacity will decrease to less than about 70%, preferably less thanabout 50% and more preferably less than about 30%, such as about 28%,after treatment with 0.5 M NaOH for 7.5 h.

In a further aspect, the present invention relates to a method ofisolating an immunoglobulin, such as IgG, IgA and/or IgM, wherein amutant protein, a multimer or a matrix according to the invention isused. Thus, the invention encompasses a process of chromatography,wherein at least one target compound is separated from a liquid byadsorption to a mutant protein or a multimer or matrix described above.The desired product can be the separated compound or the liquid. Thus,this aspect of the invention relates to affinity chromatography, whichis a widely used and well-known separation technique. In brief, in afirst step, a solution comprising the target compounds, preferablyantibodies as mentioned above, is passed over a separation matrix underconditions allowing adsorption of the target compound to ligands presenton said matrix. Such conditions are controlled e.g. by pH and/or saltconcentration i.e. ionic strength in the solution. Care should be takennot to exceed the capacity of the matrix, i.e. the flow should besufficiently slow to allow a satisfactory adsorption. In this step,other components of the solution will pass through in principleunimpeded. Optionally, the matrix is then washed, e.g. with an aqueoussolution, in order to remove retained and/or loosely bound substances.The present matrix is most advantageously used with a washing steputilising an alkaline agent, as discussed above. In a next step, asecond solution denoted an eluent is passed over the matrix underconditions that provide desorption i.e. release of the target compound.Such conditions are commonly provided by a change of the pH, the saltconcentration i.e. ionic strength, hydrophobicity etc. Various elutionschemes are known, such as gradient elution and step-wise elution.Elution can also be provided by a second solution comprising acompetitive substance, which will replace the desired antibody on thematrix. For a general review of the principles of affinitychromatography, see e.g. Wilchek, M., and Chaiken, I. 2000. An overviewof affinity chromatography. Methods Mol. Biol. 147: 1-6.

In an alternative embodiment, a mutant protein according to theinvention is used as a lead compound in a process wherein an organiccompound is modelled to resemble its three dimensional structure. The somodelled compound is known as a mimetic. Mimetic design, synthesis andtesting can be used to avoid randomly screening large number ofmolecules. In brief, such a method can involve determining theparticular parts of the protein that are critical and/or important for aproperty such as immunoglobulin-binding. Once these parts have beenidentified, its structure is modelled according to its physicalproperties, e.g. stereochemistry, bonding, size, charge etc using datafrom a range of sources, such as spectroscopic techniques, X-raydiffraction data and NMR. Computational analysis, similarity mapping andother techniques can be used in this process Important considerations inthis kind of process are the ease to synthesise a compound,pharmacological acceptance, degradation pattern in vivo etc.

Finally, the present invention also comprises other uses of the mutantprotein described above, such as in analytical methods, for medicalpurposes, e.g. for diagnosis, in arrays etc.

EXAMPLES

Below, the present invention will be described by way of examples, whichare provided for illustrative purposes only and accordingly are not tobe construed as limiting the scope of the present invention as definedby the appended claims. All references given below and elsewhere in thisapplication are hereby included herein by reference.

In this part, since Z in its original form already has a significant butnon-sufficient stability towards alkaline treatment, it was assumed thatsmall changes in stability due to the mutations would be difficult toassess in laboratory testings. Therefore, a suppressor mutation method(Kotsuka, T., S. Akanuma, M. Tomuro, A. Yamagishi, and T. Oshima. 1996.Further stabilisation of 3-isopropylmalate dehydrogenase of an extremethermophile, Thermus thermophilus, by a suppressor mutation method. J.Bacteriol. 178:723-727; and Sieber, V., A. Plückthun, and F. X. Schmidt1998. Selecting proteins with improved stability by a phage-basedmethod. Nature Biotechnology. 16:955-960) was used to provide a variantof the Z domain with a decreased structural stability. According to thisstrategy the destabilized variant of protein Z, herein denoted Z(F30A)(Cedergren et al., 1993, supra) was used as scaffold for subsequentintroduction of additional mutations related to investigations ofalkaline stability. The binding properties of this variant are similarto native protein Z, since F30 is not involved in the Fc-binding.Further, Zwt denotes the wild type Z domain, not containing the F30Asubstitution.

Experimental Strategy

To analyze which asparagines in the Z domain that are responsible forits instability in alkaline conditions, a mutational analysis wasperformed. In order to enable detection of improvements regarding thealkaline stability of the Z domain, it was decided to use a mutatedvariant, Z(F30A), since the Z-domain already possesses a significant butnon-sufficient stability towards alkaline conditions. Z(F30A) hasearlier been shown to possess an affinity to IgG that is similar to thewild type, but also a remarkably decreased structural stability due tothe mutation of an amino acid that normally takes part in thehydrophobic core (Cedergren et al., 1993, supra; Jendeberg, L., B.Persson, R. Andersson, R. Karlsson, M. Uhlén, and B. Nilsson. 1995.Kinetic analysis of the interaction between protein A domain variantsand human Fc using plasmon resonance detection. Journal of MolecularRecognition. 8:270-278). The Z-domain is a three-helix bundle consistingof 58 amino acids, including eight asparagines (N3, N6, N11, N21, N23,N28, N43 and N52) (FIG. 1) (Nilsson, B., T. Moks, B. Jansson, L.Abrahmsen, A. Elmblad, E. Holmgren, C. Henrichson, T. A. Jones, and M.Uhlén. 1987. A synthetic IgG-binding domain based on staphylococcalprotein A. Protein Eng. 1:107-113). To evaluate the effect of thedifferent asparagines on the deactivation rate in alkaline conditions,seven of these residues were exchanged for other amino acids. Since N3is located in the flexible amino-terminal of the domain, it was excludedfrom the study. It was assumed that a degradation of this amino acidwould not affect the activity of a monomeric ligand and would thereforenot be detectable in the present assay, which measures the retainedactivity. Moreover, since the amino acid is located outside thestructured part of the domain it will presumably be easily replaceableduring a multimerization of the domain to achieve a protein A-likemolecule. To facilitate the protein design, a comparison with thehomologous sequences from the other domains of protein A was made(FIG. 1) (Gülich et al., 2000a). From the comparison, it was decided toexchange asparagine 11 for a serine and 23 for threonine and finally 43for a glutamic acid. Asparagine 6 was exchanged for alanine since thealternative when looking on the homologous sequences was aspartic acid,which also has been reported to be sensitive in alkaline conditions. Allfive domains of protein A have asparagines in the other positions (21,28, 52). Hence, they were exchanged for alanines.

Example 1 Mutagenesis, Expression and Purification of Mutant Protein ZMaterials and Methods

Site-directed mutagenesis was performed using a two-step PCR-technique(Higuchi et al., 1988). Plasmid pDHZF30A (Cedergren et al., 1993) wasused as template. Oligonucleotides coding for the different asparaginereplacements and the A29G replacement were synthesised by Interactiva(Interactiva Biotechnologie GmbH, Ulm, Germany). The restriction enzymesXbaI and HindIII (MBI Fermentas Inc, Amhurst, N.Y.) were used forcloning into the vector pDHZ (Jansson et al., 1996) that was performedaccording to Sambrook (Sambrook et al., 1987). To create pTrpZ, the Zdomain was amplified by PCR, using plasmid pKN1 as template (Nord etal., 1995). The fragment was restricted with XbaI and PstI and ligatedinto the vector pTrpABDT1T2 (Kraulis et al., 1996) that had beenrestricted with same enzymes. A MEGABACE™ 1000 DNA Sequencing System(Amersham Biosciences, Uppsala, Sweden) was used to verify correctsequence of inserted fragments. MEGABACE™ terminator chemistry (AmershamBiosciences, Uppsala, Sweden) was utilised according to the supplier'srecommendations in a cycle sequencing protocol based on the dideoxymethod (Sanger et al., 1977). During cloning procedures, Escherichiacoli strain RR1ΔM15 (American Type Culture Collection, Rockville, Mass.)was used, whereas for expression of the different gene products 017(Olsson, M. O., and L. A. Isaksson. 1979. Analysis of rpsD Mutations inEscherichia coli. I: Comparison of Mutants with Various Alterations inRibosomal Protein S4. Molec. gen. Genet. 169:251-257) was used.

Production and purification of Z(F30A) and the different constructsthereof were performed according to the protocol outlined by Gülich(Gülich et al., 2000b, see above). The production of Z and pZ(N23T) wereperformed as described in Kraulis et al (Kraulis, P. J., P. Jonasson,P.-Å. Nygren, M. Uhlén, L. Jendeberg, B. Nilsson, and J. Kördel. 1996.The serum albumin-binding domain of streptococcal protein G is athree-helix bundel: a heteronuclear NMR study. FEBS lett. 378:190-194).Relevant fractions were lyophilised. The amount of protein was estimatedby absorbance measurements at 280 nm using the specific absorbancecoefficient, a (1 g⁻¹ cm⁻¹), Z 0.156; Z(N23T), 0.169; Z(F30A),Z(F30A,N43E), Z(F30A,N23T,N43E) 0.157; Z(F30A,N6A), Z(F30A,N11S),Z(F30A,N21A), Z(F30A,N23T), Z(F30A,N28A), Z(F30A,N52A),Z(F30A,N6A,N23T), Z(F30A,N11S,N23T) 0.158. The concentration wasconfirmed by amino acid analysis (BMC, Uppsala, Sweden). The homogeneitywas analysed by Sodium Dodecyl Sulfate PolyAcrylamide GelElectrophoresis (SDS-PAGE) (Laemmli, U.K. 1970. Cleavage of StructuralProteins during the Assembly of the Head of Bacteriophage T4. Nature.227:680-685) using the Phast system. Lyophilised proteins were loaded onhigh-density gels (Amersham Biosciences, Uppsala, Sweden) under reducingconditions and stained with Coomassie Brilliant Blue according to thesupplier's recommendations. The homogeneity and the molecular weightswere further confirmed by mass spectrometry.

For CD spectroscopy, protein samples were prepared in a phosphate buffer(8.1 mM K₂HPO₄, 1.9 mM KH₂PO₄, pH 7.5) to a concentration of 10 μM.Spectra were recorded using a J-720 spectropolarimeter (JASCO, Tokyo,Japan) in the far UV region from 250 to 200 nm at RT in a quartz cell ofpath length 0.1 cm and with a scan speed of 10 nm min⁻¹. Each spectrumwas the mean of five accumulated scans and the final spectra wereconverted into mean residue ellipticity (MRE) (deg cm² dmol⁻¹).

Results Example 1

All Z variants were successfully produced intracellular in E. coli at37° C. and show the same expression levels, approximately 50 mg/l asestimated from SDS-PAGE. The proteins were all purified by IgG affinitychromatography. After the purification, samples were analysed withSDS-PAGE (data not shown), lyophilised and stored for further analyses.The molecular mass for protein Z and the different mutants thereof werealso confirmed by mass spectrometry. The data confirmed correct aminoacid content for all mutants (data not shown). Also, structural analyseswere performed on a Circular Dichroism (CD) equipment, since itpreviously has been proven to be suitable for detecting structuralchanges in α-helical proteins (Johnson, C. W., Jr. 1990. Proteinsecondary structure and circular dichroism: a practical guide. Proteins.7:205-214; and Nord, K., J. Nilsson, B. Nilsson, M. Uhlén, and P.-Å.Nygren. 1995. A combinatorial library of an a-helical bacterial receptordomain. Prot. eng. 8:601-608). All spectra show a minimum at 208 nm andat 222 nm in combination with a maximum around 195 nm, indicating asimilar structure for the mutants and the parental molecule. However,Z(F30A,N52A) seems to have a somewhat lower α-helicity than the wildtype Z and the other mutants thereof (data not shown).

Example 2 Biospecific Interaction Analysis Materials and Methods

Differences in affinity and kinetic constants of the association anddissociation states were detected on a BIACORE™ 2000 instrument(Biacore, Uppsala, Sweden). Human polyclonal IgG and HSA (negativereference) were immobilised by amine coupling on the carboxylateddextran layer of a CM5 sensor chip (BIACORE™) according to thesupplier's recommendations. The immobilisation of IgG resulted inapproximately 2000 RU. Z, ZF30A, and the different mutants were preparedin HBS (10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH7.4) at 10 different concentrations (100-550 nM). The samples wereinjected over the surfaces as duplicates in random order at a flow rateof 30 μl min⁻¹. 10 mM HCl was used to regenerate the surface. The datawas analysed using the BIA evaluation 3.0.2b software (Biacore AB). Thesignals from a control surface immobilized with HSA were subtracted fromthe IgG surface. A 1:1 Langmuir model was assumed and apparent kineticconstants and also affinity constants were calculated. Also, the changein free binding energy (ΔΔG=−RTln K_(aff, mutant)/K_(aff, native)) inrelation to the native molecule was calculated.

Results Example 2

To determine the differences in affinity for the Z variants towards IgG,surface plasmon resonance (SPR) using a BIACORE™ was carried out. Theaim was to compare the affinity for the different mutated Z variantsaccording to the invention with the parental molecule. As mentionedabove, due to the high alkaline stability of the parental Z domain itwas decided to use a structurally destabilized variant of Z includingthe F30A mutation (Cedergren, L., R. Andersson, B. Jansson, M. Uhlén,and B. Nilsson. 1993. Mutational analysis of the interaction betweenstaphylococcal protein A and human IgG₁ . Protein eng. 6:441-448).Therefore, it was of importance to first confirm that the affinitybetween the mutated molecule and IgG was retained despite the mutation.As can be seen in table 1 below, the affinity of Z(F30A) is notsignificantly affected. The very small change in affinity gives aslightly higher stability to the complex of Z(F30A) and IgG compared tothe parental molecule Z and IgG. This is in accordance with resultsearlier reported by Jendeberg et al. (Cedergren et al., 1993, supra;Jendeberg et al., 1995, supra). All mutants constructed with Z(F30A) asscaffold were analysed and compared with their parental molecule(Z(F30A)). The results show that the overall affinity is notsignificantly affected by the mutations, indicating that none of theasparagine mutations according to the invention are very important forthe binding to IgG (see table 1 below). In all Z variants including theN21A or the N43E mutation, only a slightly lower affinity constant wasobserved. For mutants with the N23T mutation, surprisingly, the affinityeven seems to be slightly higher. Also, in the case of theN28A-mutation, the decrease in affinity is very small, and cannot beexpected to have any essential influence if the mutant protein is usede.g. as a protein ligand. Furthermore, all constructs including theN28A-mutation have a remarkably increased off-rate. For the mutantsincluding the N23T mutation the somewhat increased affinity seems to bedue to a slightly increased on-rate. Also, the N6A-mutation gives ahigher on-rate, but the affinity constant is not affected because of theincreased off-rate that also follows the mutation.

TABLE 1 An overview of the kinetic study on the different Z domainscarried out using the BIACORE ™. kon koff Kaff ΔΔG (vs ΔΔG (vs [10⁵ M⁻¹[10⁻³ [110⁷ Zwt) Z(F30A)) Mutant s⁻¹] s⁻¹] M⁻¹] [kcal/mol] [kcal/mol]Zwt 1.5 3.7 4.0 0 Z(N23T) 2.7 3.9 7 −0.3 Z(F30A) 1.9 4.17 4.5 −0.1 0.0Z(F30A, N6A) 7 21 3.3 0.1 0.2 Z(F30A, N11S) 1.6 4.9 3.2 0.1 0.2 Z(F30A,N21A) 1 3.8 2.6 0.3 0.4 Z(F30A, N23T) 2.1 3.75 5.6 −0.2 −0.1 Z(F30A,N28A) 3.1 9.87 3.2 0.1 0.2 Z(F30A, N43E) 1.3 5.1 2.6 0.3 0.4 Z(F30A,N52A) 1.5 4.9 3 0.2 0.3 Z(F30A, N23T, 0.8 3.8 2 0.4 0.5 N43E)

Z_(wt) was used as an internal standard during the differentmeasurements. The differences in free binding energy are calculatedrelative to Zwt and Z(F30A) respectively.

Example 3 Stability Towards Alkaline Conditions Materials and Methods

The behaviour of the variants of domain Z as affinity ligands wasanalysed by immobilisation to a standard affinity matrix. Z, Z(F30A),and mutated variants were covalently coupled to HITRAPT™ affinitycolumns (Amersham Biosciences, Uppsala, Sweden) using theN-hydroxysuccinimide chemistry according to the manufacturer'srecommendations. The columns were pulsed with TST and 0.2 M HAc, pH 3.1.Human polyclonal IgG in TST was prepared and injected onto the columnsin excess.

A standard affinity chromatography protocol was followed for 16 cycleson the ÄKTA™explorer 10 (Amersham Biosciences, Uppsala, Sweden). Betweeneach cycle a CIP-step was integrated. The cleaning agent was 0.5 M NaOHand the contact time for each pulse was 30 minutes, resulting in a totalexposure time of 7.5 hours. Eluted material was detected at 280 nm.

Results Example 3

Z, Z(F30A), and mutants thereof were covalently attached to HITRAP™columns using NHS-chemistry. IgG in excess was loaded and the amount ofeluted IgG was measured after each cycle to determine the total capacityof the column. Between each cycle the columns were exposed to CIPtreatment consisting of 0.5 M NaOH. After 16 pulses, giving a totalexposure time of 7.5 hours, the column with the Z(F30A)-matrix shows a70% decrease of the capacity. The degradation data in FIG. 2 a suggestthat four of the exchanged asparagines (N6, N11, N43 and N52) are lesssensitive to the alkaline conditions the mutants are exposed for in thisexperiment. In contrast, N23 seems to be very important for thestability of Z(F30A). Z(F30A,N23T) shows only a 28% decrease of capacitydespite the destabilizing F30A-mutation. Hence, the Z(F30A,N23T) isalmost as stable as Zwt and thereby the most stabilised variant withZ(F30A) as scaffold. Also the Z(F30A)-domain with two additionalmutations Z(F30A,N23T,N43E) shows the same pattern of degradation asZ(F30A,N23T). An exchange of N28 to an alanine also improves thestability of Z(F30A) towards alkaline conditions. Surprisingly, thecolumn with Z(F30A,N21A) as affinity ligand reveals a dramatic loss ofcapacity when exposed to NaOH compared to the parental molecule. Thesedata make Z(N23T) to a very advantageous candidate as ligand in affinitypurification of IgG.

To finally prove the reliability of the strategy using a structurallydestabilized variant of a molecule in order to make small changes instability detectable, the N23T-mutation was grafted into the parentalZ-domain. Both the parental Z-domain and Z(N23T) were coupled toHITRAP™-columns and exposed to alkaline conditions in the same way asfor the already mentioned mutants. As can be seen in FIG. 2 b, theZ(N23T)-mutant shows higher stability than Zwt when exposed to high pH.

Example 4 Construction of Monomers of Z-Mutants with and without aC-Terminal Cysteine

Three different mutations were introduced in a gene encoding Z(N23T):

K4G, N3A and the double-mutation N3A/N6D.

The mutations were originally introduced into two different vectors: onewith a cysteine in the C-terminus and one without the cysteine. This wasdone to later facilitate the construction of multimers with one singleC-terminal cysteine.

Example 4(a) Cysteine-Containing Monomer Construction

As template for the construction, a plasmid denoted “pGEM ZN23T”, wasused. This already contained the N23T-mutation in the Z-gene.

A PCR-reaction was performed with this plasmid as template and the twooligonucleotides

 (SEQ ID NOs: 24-25) AFFI-63: TTT TTT GTA GAC AAC GGA TTC AAC AAA GAA CGRTO-40: GAT CTG CTG CAG TTA GCA TTT CGG CGC CTG AGC ATC ATT TAGfor the K4G-mutation, (SEQ ID NOs: 26-27) AFFI-64:TTT TTT GTA GAC GCC AAA TTC AAC AAA GAA C GRTO-40:GAT CTG CTG CAG TTA GCA TTT CGG CGC CTG AGC ATC ATT TAGfor the N3A-mutation and, (SEQ ID NOs: 28-29) AFFI-65:TTT TTT GTA GAC GCC AAA TTC GAC AAA GAA C GRTO-40:GAT CTG CTG CAG TTA GCA TTT CGG CGC CTG AGC ATC ATT TAGfor the N3A/N6D-mutation.

PCR reaction tubes containing: 0.5 μl template pGEM ZN23T [500 ng/μl], 5pmol of each primer (Interactiva, Thermo Hybaid GmbH, Ulm, Germany), 5μl of dNTP-mix ([10 mM], Applied Biosystems, CA, USA), 5 μl ofPCR-buffer 10× (Applied Biosystems, CA, USA), 0.1 μl of AMPLITAQ™ ([5U/μl], Applied Biosystems, CA, USA) and sterile water to a final volumeof 50 μl. The PCR-program consisted of 2 min at 94° C. followed by 30cycles of 15 sec at 96° C., 15 sec at 50° C., 1 min at 72° C. andconcluded with an additional min at 72° C. The PCR reactions wereperformed on GENEAMP® PCR System 9700 (Applied Biosystems, CA, USA).

The PCR-product was analysed on 1% agarose gel and, after confirming anobtained product of correct size, purified with QIAQUICK® PCRpurification kit (QIAGEN GmbH, Hilden, Germany).

The PCR-products were cleaved according to Sambrook (Sambrook et al.)with the restriction enzymes AccI and PstI (New England Biolabs, NEB,MA, USA). The cleavage products were analysed on agarose gel andpurified from the agarose with QIAQUICK® Gel Extraction Kit (QIAGENGmbH, Hilden, Germany) prior to ligation. The fragments were ligatedinto a vector denoted “pTrp-protA-stab-(multi9)”, already cleaved withthe enzymes AccI and PstI and purified, by adding T4 DNA ligase andligation buffer (MBI Fermentas, Lithuania), and subsequently transformedinto RRIAM15-cells (ATCC, MA, USA). The constructs were given the namespAY87 (Z(N23T/K4G)-Cys), pAY89 (Z(N23T/N3A)-Cys) and pAY91(Z(N23T/N3A/N6D)-Cys), respectively.

A MEGABACE™ 1000 DNA Sequencing System (Amersham Biosciences, Uppsala,Sweden) was used to verify correct sequences of inserted fragments.MEGABACE™ terminator chemistry (Amersham Biosciences, Uppsala, Sweden)was utilised according to the supplier's recommendations in a cyclesequencing protocol based on the dideoxy method (Sanger et al., 1977).

Example 4(b) Non-Cysteine-Containing Monomer Construction

As template for the construction, a plasmid denoted “pTrp(—N)ZN23T-Cys”,was used. This plasmid already contained the gene with theN23T-mutation.

A PCR-reaction was performed with this plasmid as template and the twooligonucleotides

(SEQ ID NOs: 30-31) AFFI-63: TTT TTT GTA GAC AAC GGA TTC AAC AAA GAA CGRTO-41: GAT CTC GTC TAC TTT CGG CGC CTG AGC ATC ATT TAGfor the K4G-mutation,  (SEQ ID NOs: 32-33) AFFI-64:TTT TTT GTA GAC GCC AAA TTC AAC AAA GAA C GRTO-41:GAT CTC GTC TAC TTT CGG CGC CTG AGC ATC ATT TAG for the N3A-mutation,(SEQ ID NOs: 34-35) AFFI-65: TTT TTT GTA GAC AAC GGA TTC AAC AAA GAA CGRTO-41: GAT CTC GTC TAC TTT CGG CGC CTG AGC ATC ATT TAGand for the N3A/N6D-mutation.

PCR-reaction tubes containing: 0.5 μl template pTrp(—N)ZN23T-Cys [500ng/μl], 5 pmol of each primer (Interactiva, Thermo Hybaid GmbH, Ulm,Germany), 5 μl of dNTP-mix (10 mM, Applied Biosystems, CA, USA), 5 μl ofPCR-buffer 10× (Applied Biosystems, CA, USA), 0.1 μl of AMPLITAQ™ ([5U/μl], Applied Biosystems, CA, USA) and sterile water to a final volumeof 50 μl. The PCR-program consisted of 2 min at 94° C. followed by 30cycles of 15 sec at 96° C., 15 sec at 50° C., 1 min at 72° C. andconcluded with an additional min at 72° C. The PCR reactions wereperformed on GENEAMP® PCR System 9700 (Applied Biosystems, CA, USA).

The PCR-products were directly TA-cloned into the vector pGEM accordingto the manufacturer's instructions (Promega, Wis., USA) and subsequentlytransformed into RRIΔM15-cells (ATCC, MA, USA). The constructs weregiven the names pAY86 (Z(N23T/K4G), pAY88 (Z(N23T/N3A) and pAY90(Z(N23T/N3A/N6D) respectively.

A MEGABACE™ 1000 DNA Sequencing System (Amersham Biosciences, Uppsala,Sweden) was used to verify correct sequences of inserted fragments.MEGABACE™ terminator chemistry (Amersham Biosciences, Uppsala, Sweden)was utilised according to the supplier's recommendations in a cyclesequencing protocol based on the dideoxy method (Sanger et al., 1977).

Example 5 Construction of the Gene Encoding Monomers and Oligomers witha C-Terminal Cysteine in pTrp-Vector

All of the above described plasmids pAY 86 to pAY91 (a total of sixplasmids) were cleaved with the restriction enzyme AccI. This resultedin releasing the Z-mutants completely from the pAY86-, pAY88- andpAY90-vectors and a single opening at the 3′-end of the gene in thepAY87-, pAY89- and pAY91-vectors.

The cleaved vectors were treated with Calf Intestine AlkalinePhosphatase (CIAP, MBI Fermentas, Lithuania) according to themanufacturer's recommendations. This step was performed todephosphorylate the 5′-ends to avoid self-ligation of the vectors.

The released Z-mutant-fragments were analysed on agarose gel andsubsequently purified from the agarose before the fragments was ligatedinto the opened vectors according to the following:

fragment from pAY86 to pAY87fragment from pAY88 to pAY89fragment from pAY90 to pAY91

For the ligation reactions, different proportions of fragment versusvector were mixed and the result was that a range of differentmultimers, as expected, ranging from dimers to pentamers was obtained.

The different multimers were transformed into RRIΔM15-cells (ATCC, MA,USA) and the correct sequences were verified by analysis on a sequencingequipment at the Royal Institute of Technology as described above. Thenewly constructed plasmids were denoted as shown in the table below:

TABLE 2 Summary of constructed plasmids Plasmid no. Expressed proteinpAY from construct 86 Z(N23T/K4G) 87 Z(N23T/K4G)-Cys 88 Z(N23T/N3A) 89Z(N23T/N3A)-Cys 90 Z(N23T/N3A/N6D) 91 Z(N23T/N3A/N6D)-Cys 92Z(N23T/K4G)dimer-Cys 93 Z(N23T/N3A)dimer-Cys 94 Z(N23T/N3A/N6D)dimer-Cys95 Z(N23T/K4G)trimer-Cys 96 Z(N23T/N3A)trimer-Cys 97Z(N23T/N3A/N6D)trimer-Cys 98 Z(N23T/K4G)tetramer-Cys 99Z(N23T/N3A)tetramer-Cys 100 Z(N23T/N3A/N6D)tetramer-Cys 101Z(N23T/K4G)pentamer-Cys 102 Z(N23T/N3A)pentamer-Cys 103Z(N23T/N3A/N6D)pentamer-Cys

The above described plasmid vectors, except pAY86, pAY88 and pAY90 haveTrp promoter, Trp leader sequence and a gene for kanamycin (Km)resistance. pAY86, pAY88 and pAY90 have a gene for ampicillin resistanceinstead.

Example 6 Construction of Genes Encoding Monomers and Oligomers with aC-Terminal Cysteine in pK4-Vector

The genes encoding the proteins as summarized in Table 2 were to betransferred to a vector containing SPA promoter and signal sequence. Toenable this procedure, an adapter containing the cleavage site for therestriction enzyme KpnI (New England Biolabs, NEB, MA, USA) was to beconstructed. The adapter was constructed by the two oligonucleotides(Interactiva, Thermo Hybaid GmbH, Ulm, Germany)

The plasmid pAY104 (pK4-cys-ABDstabdimer) was cleaved with FspI and PstI(New England Biolabs, NEB, MA, USA). The vector was purified on agarosegel and the released fragment was removed and the remaining vector waspurified from the agarose with QIAQUICK® Gel Extraction Kit (QIAGENGmbH, Hilden, Germany).

The two oligomers AFFI-88 and AFFI-89 were mixed in ligation buffer (MBIFermentas, Lithuania) and heated to 50° C. and the mixture was allowedto cool to room temperature where after the cleaved plasmid vector wasadded together with T4 DNA ligase (MBI Fermentas, Lithuania). After theligation reaction, the product was transformed into RRIΔM15-cells andthe correct sequence was verified as described above. The resultingplasmid was denoted pAY128.

The plasmid pAY128 was then cleaved with the restriction enzymes KpnIand PstI and the cleaved vector was analysed on agarose gel andsubsequently purified from the agarose with QIAQUICK® Gel Extraction Kit(QIAGEN GmbH, Hilden, Germany). The fragments expressing the two mutatedZ-genes Z(N23T/N3A) and Z(N23T/N3A/N6A) from pAY86 to pAY103 werecleaved with KpnI and PstI (New England Biolabs, NEB, MA, USA),separated and purified after agarose gel separation. The differentfragments were ligated into the cleaved vector originating from pAY128and the resulting plasmids were, after verifying correct sequences,denoted pAY107 to pAY116 as summarized in Table 3.

TABLE 3 Summary of constructed plasmids with SPA promoter and signalsequence. Plasmid no. Expressed protein pAY from construct 107Z(N23T/N3A)-Cys 108 Z(N23T/N3A/N6D)-Cys 109 Z(N23T/N3A)dimer-Cys 110Z(N23T/N3A/N6D)dimer-Cys 111 Z(N23T/N3A)trimer-Cys 112Z(N23T/N3A/N6D)trimer-Cys 113 Z(N23T/N3A)tetramer-Cys 114Z(N23T/N3A/N6D)tetramer-Cys 115 Z(N23T/N3A)pentamer-Cys 116Z(N23T/N3A/N6D)pentamer-Cys

Example 7 Construction of Genes Encoding a Part of the E-Gene (E′) fromProtein a N-Terminally Fused to Monomers and Oligomers with a C-TerminalCysteine in pK4-Vector

The genes encoding the proteins, as summarized in Table 2, weretransferred to a vector containing the SPA promoter and signal sequenceand a part of the gene encoding the E-region of protein A (E′). It hasearlier been shown that an addition of the N-terminal IgG-binding partof the mature protein A (region E), or parts thereof, may increasecorrect processing and also facilitate secretion of the gene product tothe surrounding culture medium (Abrahmsén et al., 1985). An adaptercontaining the cleavage site for the restriction enzyme KpnI and a partof region E from protein A (E′) was constructed by the twooligonucleotides (Interactiva, Thermo Hybaid GmbH, Ulm, Germany)

The plasmid pAY104 (pK4-cys-ABDstabdimer) was cleaved with FspI and PstI(New England Biolabs, NEB, MA, USA). The vector was purified on agarosegel and the released fragment was removed and the remaining vector waspurified from the agarose with QIAQUICK® Gel Extraction Kit (QIAGENGmbH, Hilden, Germany). The two oligonucleotides were mixed in ligationbuffer and heated to 75° C. and the mixture was allowed to cool to roomtemperature where after the cleaved plasmid vector was added, togetherwith T4 DNA ligase (MBI Fermentas, Lithuania). After the ligationreaction the product was transformed into RRIΔM15-cells and the correctsequence was verified as described above. The resulting plasmid wasdenoted pAY129.

The plasmid pAY129 was then cleaved with the restriction enzymes KpnIand PstI and the cleaved vector was analysed on agarose gel andsubsequently purified from the agarose with QIAQUICK® Gel Extraction Kit(QIAGEN GmbH, Hilden, Germany). The fragments expressing the two mutatedZ-genes Z(N23T/N3A) and Z(N23T/N3A/N6A) from pAY86 to pAY103 werecleaved with KpnI and PstI, separated and purified after agarose gelseparation. The different fragments were ligated into the cleaved vectororiginating from pAY129 and the resulting plasmids were, after verifyingcorrect sequences, denoted pAY118 to pAY127 as summarized in Table 4.

TABLE 4 Summary of constructed plasmids with SPA promoter and signalsequence and a part of region E from protein A-E′. Plasmid no. Expressedprotein pAY from construct 118 E′-Z(N23T/N3A)-Cys 119E′-Z(N23T/N3A/N6D)-Cys 120 E′-Z(N23T/N3A)dimer-Cys 121E′-Z(N23T/N3A/N6D)dimer-Cys 122 E′-Z(N23T/N3A)trimer-Cys 123E′-Z(N23T/N3A/N6D)trimer-Cys 124 E′-Z(N23T/N3A)tetramer-Cys 125E′-Z(N23T/N3A/N6D)tetramer-Cys 126 E′-Z(N23T/N3A)pentamer-Cys 127E′-Z(N23T/N3A/N6D)pentamer-Cys

Example 8 Stability Towards Alkaline Conditions

To evaluate the stability of the proteins towards alkaline conditions,four different proteins were tested in an environment of high pH. Thedifferent proteins were Z(N23T)dimer-Cys, Z(N23T/K4G)dimer-Cys,Z(N23T/N3A)dimer-Cys and Z(N23T/N3A/N6D)dimer-Cys.

(Z(N23T)dimer-Cys), (Z(N23T/N3A)dimer-Cys), (Z(N23T/N3A/N6D)dimer-Cys)and (Z(N23T/K4G)dimer-Cys) were cultivated in fermenters. The harvestedmedia were purified and coupled to HF Agarose (Amersham Biosciences,Uppsala, Sweden) using standard methods before the alkaline tests. TheHF agarose-coupled proteins were denoted as follows

Z(N23T)dimer-Cys U631049 Z(N23T/K4G)dimer-Cys U631079Z(N23T/N3A)dimer-Cys U631064 Z(N23T/N3A/N6D)dimer-Cys U631063

The matrices were packed in columns (HR 5/2, Amersham Biosciences,Uppsala, Sweden) to a final volume ranging from 0.1 to 0.3 ml. Thepurification equipment used was an ÄKTA™explorer 10 (AmershamBiosciences, Uppsala, Sweden) with a UV sample flow cell with a pathlength of 2 mm (Amersham Biosciences, Uppsala, Sweden).

The buffers used contained

-   -   Running buffer: 25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05%        Tween 20, 5 mM ammonium acetate, pH 8.0    -   Elution buffer: 0.2 M acetic acid (HAc), pH 3.1    -   Cleaning-In-Place (CIP) buffer: 0.5 M NaOH

A typical chromatographic run cycle consisted of

-   -   Equilibrium of the column with running buffer    -   Sample application of 10 mg polyclonal human IgG (hIgG) at 0.2        ml/min    -   Extensive washing-out of unbound proteins    -   Elution at 1.0 ml/min with elution buffer    -   Re-equilibration with running buffer    -   Cleaning-In-Place (CIP) with CIP-buffer with a contact time        between column matrix and 0.5 M NaOH of 1 hour    -   Re-equilibration with running buffer

The amount of hIgG loaded at each run was well above the total dynamicbinding capacity of the column since the breakthrough of unbound proteinwas considerable when loading the sample onto the columns in all cases.

After one cycle, including the steps above, a new cycle was startedwhich again included one hour of exposure of 0.5 M sodium hydroxide. Tomeasure the decrease of the dynamic binding capacity of the column thepeak area of the eluted peak was compared with the original peak area ofthe eluted peak when the matrix had not been exposed to the sodiumhydroxide. Setting the original peak area as 100% of binding capacitythe decrease of the binding capacity of hIgG was observed. The peak areawas calculated with the UNICORN™ software accompanying the purificationsystem.

Each cycle was repeated 21 times resulting in a total exposure timebetween the matrix and the sodium hydroxide of 20 hours for eachdifferent matrix. The normalized peak areas were visualised in a graphas can be seen below (FIG. 16). All 21 cycles were repeated for eachmutant.

Both Z(N23T/N3A/N6D)dimer-Cys and Z(N23T/N3A)dimer-Cys showed improvedstability against alkaline conditions compared to the originallyproduced Z(N23T)dimer-Cys.

Example 9 Preparation of an Fc-Binding Affinity Ligand Materials Samples

-   HERCEPTIN®: HERCEPTIN® 50 mg    -   powder to concentrate to    -   infusion liquid, solution    -   Trastuzumab    -   Roche    -   Lot B1171    -   EU/1/00/145/001    -   Roche Registration Limited    -   40 Broadwater Road    -   Welwyn Garden City    -   Hertfordshire, AL7 3AY    -   UK-   ENBREL™: ENBREL™ 25 mg    -   powder to injection liquid, solution    -   etanercept    -   Wyeth    -   Lot 18028    -   EU/1/99/126/003    -   Wyeth Europe Ltd.,    -   Huntercombe Lane South,    -   Taplow, Maidenhead,    -   Berkshire,    -   SL6 OPH,    -   UK-   SYNAGIS®: SYNAGIS® 100 mg    -   Palivizumab    -   powder and liquid to    -   injection liquid, solution    -   Abbott    -   Lot 28423TFX    -   EU/1/99/117//002    -   Abbott Laboratories Ltd.    -   Queenborough    -   Kent ME11 5EL    -   UK

Columns

The columns used in this example were as presented in Table 5 below:

TABLE 5 Name in report Ligand Column Batch Zwt (Zwt)4 Column 2 1555047BMABSELECT ™ MABSELECT ™ Column 2 U1555045A Bwt (Bwt)4 Column 1 U1555051BSURE ™ Alkali-stabilized Column 6 U669082 Domain B SURE ™Alkali-stabilized Column 2 U669082 Domain B

Zwt refers to Protein Z, which is a mutated Domain B. It is denoted Zwtfor ‘wild type’ herein, to clarify that the only mutation it comprisesis that from Domain B to Protein Z. Protein Z has been described e.g. inU.S. Pat. No. 5,143,844.

MABSELECT™ refers to the commercially available product (GE Healthcare,Uppsala, Sweden), wherein the ligands are comprised of recombinantlyproduced Protein A, which has not been mutated to improve the stabilityunder alkaline conditions.

Bwt refers to the true wild type of Domain B of SpA.

SURE™ refers to the alkali-stable Domain B of SpA (SURE™) preparedaccording to the present invention, as described in the precedingexamples.

Each column was packed with 2 ml matrix.

SURE™ Column 6 was used for both HERCEPTIN® runs in Experiment 1 and oneof the ENBREL™ runs in Experiment 1 (see Method description below).

SURE™ Column 2 was used in the rest of the runs.

Reagents and Chemicals

-   Citric acid: Citric Acid Monohydrate, Merck, 1.00244.0500, lot    K91294344538-   NaCl: NaCl, Scharlau, SO 0227, lot 75078-   NaOH: Sodium hydroxide solution 50%, Merck, 1.58793.1000, lot    B612893517-   Water: MILLI-Q™-   Sterile water: Autoclaved MILLI-Q™-   pH 7 standard: Buffer solution pH=7.00 (20° C.), yellow-coloured,    Scharlau SO2007, Batch 73997-   pH 2 standard: Buffer solution pH=2 (Citric acid/Sodium    hydroxide/Hydrochloric acid), Scharlau 501022, Batch 72053

Buffers and Solutions

-   Buffer A: 50 mM Citric acid, 0.15 M NaCl, pH 6.0-   Buffer B: 50 mM Citric acid, 0.15 M NaCl, pH 2.5

Instruments

-   Chromatography system: ÄKTA™explorer 10 controlled by the software    UNICORN™ 5.01, GE Healthcare-   Column hardware: TRICORN™ 5/100 GL, GE Healthcare-   Sample application device: SUPERLOOP™ (50 ml), GE Healthcare-   pH meter: Laboratory pH Meter CG 842, SCHOTT-   Filter for buffer: 75 mm Bottle Top Filter-500 ml, 0.2 μm pore size,    Nalgene-   Filter for sample: MINISART® Single use syringe filter, 0.2 μm pore    size, Sartorius

Methods Buffer Preparation

Citric acid and NaCl were dissolved in water. A pH meter was calibratedusing pH 7 and pH 2 standard buffers. The pH was monitored while addingNaOH to the buffers until pH reached 6 and 2.5 for Buffer A and Buffer Brespectively. The buffers were degassed and filtered prior use.

Sample Preparation Preparation of HERCEPTIN®

HERCEPTIN® was dissolved to 21 mg/ml according to the manufacturersinstructions by adding 7.2 ml sterile water into a vial containinglyophilized Trastuzumab (HERCEPTIN®).

For Experiment 1a 1 mg/ml solution of HERCEPTIN® was prepared bydiluting the 21 mg/ml solution with Buffer A.

For Experiment 2 a 5 mg/ml solution of HERCEPTIN® was prepared bydiluting the 21 mg/ml solution with Buffer A.

Preparation of ENBREL™

ENBREL™ was dissolved to 25 mg/ml according to the manufacturersinstructions by adding 1 ml water for injection (provided by themanufacturer) into a vial containing lyophilized etanercept (ENBREL™)

For Experiment 1a 1 mg/ml solution of ENBREL™ was prepared by dilutingthe 25 mg/ml solution with Buffer A.

For Experiment 2 a 2.5 mg/ml solution of ENBREL™ was prepared bydiluting the 25 mg/ml solution with Buffer A.

Preparation of SYNAGIS®

SYNAGIS® was dissolved to 100 mg/ml according to the manufacturersinstructions by adding 1 ml water for injection (provided by themanufacturer) into a vial containing lyophilized Palivizumab (SYNAGIS®).

For Experiment 1 a 1 mg/ml solution of SYNAGIS® was prepared by dilutingthe 100 mg/ml solution with Buffer A. The 1 mg/ml solution was filteredprior sample application due to low levels of precipitation formed upondilution.

For Experiment 2 a 5 mg/ml solution of SYNAGIS® was prepared by dilutingthe 100 mg/ml solution with Buffer A. The 5 mg/ml solution was filteredprior sample application due to low levels of precipitation formed upondilution.

Method Description

This report describes two experiments—Experiment 1 and Experiment 2.

In Experiment 1 three biopharmaceuticals (HERCEPTIN®, ENBREL™ andSYNAGIS®) were loaded on and eluted from four different columns (packedwith Zwt, MABSELECT™, Bwt and SURE™)

In Experiment 2 a higher load of the three biopharmaceuticals wasapplied onto two columns packed with MABSELECT™ and SURE™.

Experiment 1

Each “run” in Experiment 1 was a scouting in which one type of samplewas run on four different columns Two such runs were performed on eachsample and three different samples were used. This makes 4×2×3=24individual runs i.e. chromatograms.

The following text describes a typical run. Four different columns wereattached to ÄKTA™explorer 10. A 1 mg/ml solution of one type of sample(HERCEPTIN®, ENBREL™ or SYNAGIS®) was injected into a SUPERLOOP™. Fourcollection tubes were connected to four positions of the outlet valve.Buffer tubing from the A pump was placed in Buffer A and buffer tubingfrom the B pump was placed in Buffer B. The ÄKTA™explorer pH electrodewas calibrated with pH 7 and pH 2 standard buffers. A 1 cm UV cell wasused.

ÄKTA™explorer 10 was controlled by UNICORN™ and schematically theprogram, or method, for Experiment 1 consisted of following parts:

-   Equilibration: 3 column volumes (CV) equilibration of the column    with Buffer A.-   Sample loading: 2 ml 1 mg/ml sample loaded onto the column (1 mg    sample per ml matrix).-   Wash: 4 CV wash of the column with Buffer A.-   Elution: 20 CV gradient from 0% Buffer B to 100% Buffer B. After the    gradient the column was washed with 5 additional column volumes of    100% Buffer B.-   Reequilibration: 3 CV reequilibration with Buffer A.

In the elution step a watch function was included in the method: whenthe absorbance at 280 nm reached 50 mAu this watch was activated andmade the outlet valve to switch to a specified position, enabling thecollection of an eluted peak in a collection tube. When the absorbanceat 280 nm dropped below 50 mAu the outlet valve switched back to itsdefault waste position. Since a scouting run included four differentcolumns the eluted peaks were collected in four individual tubes.Collecting peaks this way made it possible to measure pH of the peaki.e. the pH at which the sample was eluted. pH values of the eluateswere measured with a laboratory pH meter calibrated with pH 7 and pH 2standard buffers. pH was also monitored during the run with the ÄKTA™ pHmeter.

Experiment 2

In Experiment 2 a higher amount of sample was applied to the columnsThis was done to achieve conditions similar to those used in Protein Achromatography processes. Experiment 1 and Experiment 2 were performedessentially in the same way. The differences are listed below:

-   Columns: Two columns were used in this experiment—MABSELECT™ and    SURE™-   Number of runs: Single runs were performed (i.e. two columns, one    run per sample and three different samples makes six individual    runs, or chromatograms).-   Sample: 8 ml 5 mg/ml HERCEPTIN® (20 mg sample per ml matrix) 7 ml    2.5 mg/ml ENBREL™ (8.75 mg sample per ml matrix) 8 ml 5 mg/ml    SYNAGIS® (20 mg sample per ml matrix)-   UV cell: A 0.2 cm UV cell was used. This cell is less sensitive and    more appropriate for larger amounts of sample.-   Watch: The peak collection watch in the gradient was set to 100 mAU.

Evaluation of Chromatographic Results

pH of the eluted peaks was measured with an external pH meter after eachscouting run. These pH values are called “External pH on eluate” in 3.1.pH measurements with the ÄKTA™ pH electrode could not be used in theevaluation of the results in the most desired way due to a drift of thepH curve over time. However, one pH curve—the curve that best matchedthe pH of the buffers (pH 6 and 2.5)—was used for all chromatograms whenmeasuring the pH at which the eluted peak had its maximum absorbance at280 nm (called “ÄKTA™ pH at max UV” in 3.1).

UV₂₈₀ curves from the chromatographic runs were overlaid and grouped bysample. Thus, in Experiment 1 there are three figures for three samples(HERCEPTIN®, ENBREL™ and SYNAGIS®). Each figure displays two curves fromfour columns (Zwt, MABSELECT™, Bwt and SURE™). In Experiment 2 there arethree figures for three samples (HERCEPTIN®, ENBREL™ and SYNAGIS®). Eachfigure displays one curve from two columns (MABSELECT™ and SURE™)

Results Experiment 1

TABLE 6 ÄKTA ™ pH External pH Sample Ligand at max UV on eluateHERCEPTIN ® Zwt 3.60 3.60 HERCEPTIN ® Zwt 3.61 3.63 HERCEPTIN ®MABSELECT ™ 3.14 3.17 HERCEPTIN ® MABSELECT ™ 3.13 3.15 HERCEPTIN ® Bwt3.14 3.18 HERCEPTIN ® Bwt 3.14 3.18 HERCEPTIN ® SURE ™ 3.65 3.68HERCEPTIN ® SURE ™ 3.64 3.67

TABLE 7 ÄKTA ™ pH External pH Sample Ligand at max UV on eluate ENBREL ™Zwt 3.76 3.82 ENBREL ™ Zwt 3.76 3.84 ENBREL ™ MABSELECT ™ 3.84 3.88ENBREL ™ MABSELECT ™ 3.84 3.88 ENBREL ™ Bwt 3.79 3.83 ENBREL ™ Bwt 3.793.85 ENBREL ™ SURE ™ 3.75 3.81 ENBREL ™ SURE ™ 3.76 3.77

TABLE 8 ÄKTA ™ pH External pH Sample Ligand at max UV on eluateSYNAGIS ® Zwt 3.76 3.81 SYNAGIS ® Zwt 3.76 3.79 SYNAGIS ® MABSELECT ™3.79 3.81 SYNAGIS ® MABSELECT ™ 3.79 3.78 SYNAGIS ® Bwt 3.77 3.83SYNAGIS ® Bwt 3.76 3.79 SYNAGIS ® SURE ™ 3.76 3.77 SYNAGIS ® SURE ™ 3.763.78

Experiment 2

TABLE 9 ÄKTA ™ pH External pH Sample Ligand at max UV on eluateHERCEPTIN ® MABSELECT ™ 3.27 3.36 HERCEPTIN ® SURE ™ 3.72 3.81

TABLE 10 ÄKTA ™ pH External pH Sample Ligand at max UV on eluateENBREL ™ MABSELECT ™ 3.92 4.14 ENBREL ™ SURE ™ 3.84 4.05

TABLE 11 ÄKTA ™ pH External pH Sample Ligand at max UV on eluateSYNAGIS ® MABSELECT ™ 3.96 4.21 SYNAGIS ® SURE ™ 3.90 4.12

The above examples illustrate specific aspects of the present inventionand are not intended to limit the scope thereof in any respect andshould not be so construed. Those skilled in the art having the benefitof the teachings of the present invention as set forth above, can effectnumerous modifications thereto. These modifications are to be construedas being encompassed within the scope of the present invention as setforth in the appended claims.

1-6. (canceled)
 7. In a method of separating one or more antibodies froma liquid, which method comprises: (a) contacting said liquid with aseparation matrix comprising ligands immobilised to a support; (b)allowing antibodies to adsorb to said matrix by interaction with saidligands; (c) optionally washing the adsorbed antibodies; and (d)recovering antibodies by contacting said matrix with an eluent whichreleases the antibodies; the improvement being that said ligandscomprise one or more alkali-stable domain B of staphylococcal Protein A(SpA) wherein at least one glycine has been replaced by an alanine. 8.The method of claim 7, wherein the recovery of antibodies is achieved byadding an eluent having a pH in the range of 3.8-3.9.
 9. The method ofclaim 7, wherein at least 80% of the antibodies are recovered using aneluent having a pH in the range of 3.7-3.9.
 10. The method of claim 7,wherein at least 90% of the antibodies are recovered using an eluenthaving a pH in the range of 3.7-3.9.
 11. The method of claim 7, whereinat least 95% of the antibodies are recovered using an eluent having a pHin the range of 3.7-3.9.